Method of treating eye injury with local administration of a VEGF inhibitor

ABSTRACT

Methods of reducing or treating angiogenesis and/or inflammation associated with eye injury in a subject in need thereof, comprising administering an agent capable of blocking or inhibiting vascular endothelial growth factor (VEGF) are provided. The methods are useful for inhibiting or ameliorating eye injury, particularly acute or subacute corneal injury and feature local administration (for example, subconjunctival injection or eye drops).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC § 119(e) of U.S.Provisional 60/649,232 filed 2 Feb. 2005, which application is hereinspecifically incorporated by reference in its entirety.

BACKGROUND

1. Field of the Invention

The field of the invention is related to local administration of VEGFantagonists to treat eye-related diseases, disorders and injuries.

2. Description of Related Art

It has previously been reported that topical application of an anti-VEGFneutralizing antibody suppresses acute allograft rejection in a ratcorneal transplant model (Yatoh et al. (1998) Transplantation66(11):1519-24).

BRIEF SUMMARY OF THE INVENTION

The invention is based in part on the finding that local administrationof an agent capable of blocking, inhibiting, or reducing the activity ofvascular endothelial growth factor (VEGF) is useful in treating ofangiogenesis and inflammation associated with eye injuries or infection.

In a first aspect, the invention features a method of treating an eyeinjury, comprising locally administering an effective amount of an agentcapable of blocking or inhibiting vascular endothelial growth factor(VEGF)-mediated activity to a subject in need thereof, such that the eyeinjury is ameliorated or improved. Preferably, the eye injury is acorneal injury or conjunctival injury and the method of treatmentreduces angiogenesis and inflammation associated with the eye injury.

In specific embodiments, the agent capable of blocking, inhibiting, orameliorating VEGF-mediated activity is a VEGF antagonist comprising afusion polypeptide selected from the group consisting of acetylatedFlt-1(1-3)-Fc, Flt-1(1-3_(R→N))-Fc, Flt-1(1-3_(ΔB))-Fc,Flt-1(2-3_(ΔB))-Fc, Flt-1(2-3)-Fc, Flt-1D2-VEGFR3D3-FcΔC1(a),Flt-1D2-Flk-1D3-FcΔC1(a), and VEGFR1R2-FcΔC1(a). In a specific andpreferred embodiment, the VEGF trap is VEGFR1R2-FcΔC1(a) (also termedVEGF trap_(R1R2)) comprising the nucleotide sequence set forth in SEQ IDNO: 1 and the amino acid sequence set forth in SEQ ID NO: 2. Theinvention comprises the use of a VEGF trap that is at least 90%, 95%,98%, or at least 99% homologous with the nucleotide sequence set forthin SEQ ID NO: 1 and/or the amino acid sequence set forth in SEQ ID NO:2.

The method of the invention is useful to treat acute and sub-acutecorneal injury or conjunctival injury. Acute corneal injury may betreated within 24 hours of occurrence, and includes corneal injury orconjunctival injury caused by a penetrating object, a foreign body, or achemical or burn injury. A sub-acute injury may be treated up to twoweeks post-injury and may include the above listed injuries as well asinfectious etiologies.

In various embodiments, the eye injury is caused by trauma, e.g.,surgical injuries, chemical burn, corneal transplant, infectious orinflammatory diseases.

Length of treatment will vary according to the injury, but treatmentduration may be short, e.g., up to one month, and may include a 3-6month observation period, during which retreatment may be provided.

Administration may also include a second agent, such as animmunosuppressive agent, for example, one or more of a corticosteroid,dexamethasone, or cyclosporin A.

Local administration includes, for example, administration of the VEGFantagonist in eye drops applied to the eye, or subconjunctival injectionto the eye.

In a second aspect, the invention features a method of healing an eyeinjury, comprising locally administering an effective amount of an agentcapable of blocking or inhibiting vascular endothelial growth factor(VEGF)-mediated activity to a subject in need thereof, such that the eyeinjury heals.

In a third aspect, the invention features a method of reducing orameliorating angiogenesis associated with an eye injury, comprisinglocally administering an effective amount of an agent capable ofblocking or inhibiting vascular endothelial growth factor(VEGF)-mediated activity to a subject in need thereof, such that theangiogenesis associated with the eye injury is reduced or ameliorated.

In a fourth aspect, the invention features a method of reducing orameliorating inflammation associated with an eye injury, comprisinglocally administering an effective amount of an agent capable ofblocking or inhibiting vascular endothelial growth factor(VEGF)-mediated activity to a subject in need thereof, such that theinflammation associated with the eye injury is reduced or ameliorated.

In a fifth aspect, the invention features an ophthalmic compositioncomprising a VEGF antagonist, for example the VEGF trapVEGFR1R2-FcΔC1(a), in a pharmaceutically acceptable carrier. Suchpharmaceutical compositions may be liquid, gel, ointment, salve, slowrelease formulations or other formulations suitable for ophthalmicadministration. In various embodiments, the pharmaceutical compositionis for local administration comprising a VEGF trap, buffer, stabilizer,isotonizer, and a pharmaceutical carrier. In a preferred embodiment, thepharmaceutical composition is administered in the form of eye drops. Inspecific embodiments, the pharmaceutically acceptable carrier comprisesas least one ophthalmically acceptable excipient, wherein theophthalmically acceptable excipient can reduce a rate of removal of theVEGF antagonist from the eye by lacrimation. In various preferredembodiments, the pharmaceutical composition has an effective residencetime in the eye of about 2 to about 24 hours.

In other embodiments, the pharmaceutical composition is forsubconjunctival administration such as subconjunctival injection andsubconjunctival implantation.

In a sixth aspect, the invention features a method of administering aVEGF antagonist for treatment of angiogenesis and/or inflammationassociated with eye injury or infection, comprising local administrationby eye drops comprising a VEGF trap, or subconjunctival administrationby injection or implantation.

Other objects and advantages will become apparent from a review of theensuing detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Percent of vascularized corneal area in sutured micesubconjunctivally (SubC) treated with vehicle only or VEGF trap, atdosing regimens of (A) three 40 μg or (B) three 10 μg doses.

FIG. 2. Percent of neovascularized cornea at day 9 in sutured ratstreated SubC with vehicle only or treated with VEGF trap, with a dosingregimen of 10 μg on the day of suturing.

FIG. 3. Blood vessel length in sutured rats treated at day 0, 3 and 6with 25, 50, or 100 μg VEGF trap injections administered subcutaneously(SC) or subconjunctivally (SubC). (Control=right non-sutured eye).

FIG. 4. Quantification of blood vessel length in sutured rats receivingsubconjunctival normal saline, 5 μg, 25 μg, or 100 μg VEGF trap on day0, 3 and 6. (Control=right non-sutured eye).

FIG. 5. Quantification of corneal edema as evidenced by cornealthickness in sutured rats receiving 25 or 100 μg VEGF trap SC or normalsaline, 5 μg, 25 μg, or 100 μg VEGF trap SubC. (Control=rightnon-sutured eye) (NS=sutured eye, normal saline administered SubC).

FIG. 6. Percent reduction of edema. Effect of VEGF trap on inflammationas determined by measurement of corneal thickness. All animals weresutured (control=sutured+systemic injection (SC) of normal saline).

FIG. 7. Blood vessel length in suture-injury. Control=no suture injury.Suture control=suture+no treatment. Vehicle=suture-injury+vehicleprovided as eye drops. VEGF trap=suture-injury+1 drop three times perday (412 μg VEGF trap protein/drop).

DETAILED DESCRIPTION

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, a reference to “a method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereininclude the same meaning as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

General Description

Experiments were undertaken to evaluate corneal neovascularization aftersurgical suture placement in the cornea and to test whether cornealneovascularization following suture injury can be suppressed by localadministration of an agent capable of blocking, inhibiting, orameliorating VEGF-mediated activity. As described in the experimentalsection below, corneas of male C57BL/6 mice or Sprague-Dawley rats weresuture-injured. A molecular trap designed to inhibit VEGF-A activity wasadministered locally and tested for its ability to suppress cornealvascularization. The results revealed that sutured cornea receivingsubconjunctival administration of VEGF trap exhibited little or noneovascularization; corneal vascular area and vessel length followingsuture injury being comparable to that of a normal untreated(nonsutured) cornea. Treatment with VEGF trap eye drops following sutureinjury also effectively reduced neovascularization in suture-injuredcornea.

In addition to quantification of neovascularization as measured by anincrease in either blood vessel length or blood vessel area,suture-injury produced a marked influx of leucocytes into the injurysite. When VEGF trap was administered locally either by subconjunctivalinjection (SubC) or by eye drop, a dramatic reduction in leucocyteinfiltration was observed (data not shown).

In addition to the measurements reported below, serum levels of VEGFtrap were determined in animals treated by subconjunctival injection oreye drops of VEGF trap. As evidenced by ELISA measurement for free VEGFtrap in serum, there is little or no systemic exposure when VEGF trap isdelivered at the effective doses by either of these local (SubC or eyedrops) routes.

Definitions

The phrase “therapeutically effective dose” includes a dose thatproduces the desired effect for which it is administered. The exact dosewill depend on the purpose of the treatment, and will be ascertainableby one skilled in the art using known techniques (see, for example,Lloyd (1999) The Art, Science and Technology of PharmaceuticalCompounding).

The term “blocker”, “inhibitor”, or “antagonist” are usedinterchangeably to mean a substance that retards or prevents a chemicalor physiological reaction or response. Common blockers or inhibitorscomprise, but are not limited to, antisense molecules, antibodies,antagonists and their derivatives. More specifically, an example of aVEGF blocker or inhibitor includes a VEGF receptor-based antagonistcomprising, for example, an anti-VEGF antibody, or a VEGF trap such asVEGFR1R2-FcΔC1(a) (SEQ ID NOs:1-2).

The phrase “ophthalmically acceptable” with respect to a formulation,composition or ingredient herein means having no persistent effect thatis substantially detrimental to the treated eye or the functioningthereof, or on the general health of the subject being treated. It willbe recognized that transient effects such as minor irritation or a“stinging” sensation are common with topical ophthalmic administrationof drugs and the existence of such transient effects is not inconsistentwith the formulation, composition or ingredient in question being“ophthalmically acceptable” as herein defined. However, preferredformulations, compositions and ingredients are those that cause nosubstantial detrimental effect, even of a transient nature.

VEGF Antagonists

In various embodiments, the VEGF trap is selected from the groupconsisting of acetylated Flt-1(1-3)-Fc, Flt-1(1-3_(R→N))-Fc,Flt-1(1-3_(ΔB))-Fc, Flt-1(2-3_(ΔB))-Fc, Flt-1(2-3)-Fc,Flt-1D2-VEGFR3D3-FcΔC1(a), Flt-1D2-Flk-1D3-FcΔC1(a), andVEGFR1R2-FcΔC1(a). For a more detailed description of these and otherVEGF-receptor-based antagonists, including pegylated receptor-basedblockers, see PCT WO/00/75319, the contents of which are incorporated intheir entirety herein by reference.

In addition to the VEGF receptor-based antagonists disclosed in PCTWO/00/75319, which publication is herein specifically incorporated byreference in its entirety, variants and derivatives of such VEGFreceptor-based blockers are also contemplated by the invention. Thesequence of the variants or derivatives may differ by a change that canbe one or more additions, insertions, deletions and/or substitutions ofone or more nucleotides of the sequence set forth in SEQ ID NO:1.Changes to a nucleotide sequence may result in an amino acid change atthe protein level, or not, as determined by the genetic code. Thus,nucleic acid according to the present invention may include a sequencedifferent from the sequence shown in SEQ ID NO:1, yet encode apolypeptide with the same amino acid sequence as SEQ ID NO: 2. On theother hand, the encoded polypeptide may comprise an amino acid sequencewhich differs by one or more amino acid residues from the amino acidsequence shown in SEQ ID NO:2. A nucleic acid encoding a polypeptidewhich is an amino acid sequence variant or derivative of the sequenceshown in SEQ ID NO:2 is further provided by the present invention. Anucleic acid encoding such a polypeptide may show at the nucleotidesequence and/or encoded amino acid level greater than about 90%, 95%,98%, or 99% homology with the coding sequence shown in SEQ ID NO:1and/or the amino acid sequence shown in SEQ ID NO:2. Amino acid“homology”, may be understood to be similarity (according to theestablished principles of amino acid similarity, e.g. as determinedusing the algorithm GAP (Genetics Computer Group, Madison, Wis.)) oridentity. GAP uses the Needleman and Wunsch algorithm to align twocomplete sequences that maximizes the number of matches and minimizesthe number of gaps. Generally, the default parameters are used, with agap creation penalty=12 and gap extension penalty=4.

Individual components of the VEGF-specific fusion proteins of theinvention may be constructed by molecular biological methods known tothe art with the guidance provided by the instant specification. Thesecomponents are selected from a first cellular receptor protein, such as,for example, VEGFR1; a second cellular receptor protein, such as, forexample, VEGFR2; and a multimerizing component, such as, for example, anFc.

Specific embodiments of the VEGF-specific fusion proteins useful in themethods of the invention comprise a multimerizing component which allowsthe fusion proteins to associate, e.g., as multimers, preferably dimers.Preferably, the multimerizing component comprises animmunoglobulin-derived domain. Suitable multimerizing components aresequences encoding an immunoglobulin heavy chain hinge region (Takahashiet al. 1982 Cell 29:671-679); immunoglobulin gene sequences, andportions thereof.

The nucleic acid constructs encoding the fusion proteins useful in themethods of the invention can be inserted into an expression vector bymethods known to the art, wherein the nucleic acid molecule can beoperatively linked to an expression control sequence. Host-vectorsystems for the production of proteins comprising an expression vectorintroduced into a host cell suitable for expression of the protein areknown in the art. The suitable host cell may be a bacterial cell such asE. coli, a yeast cell, such as, for example, Pichia pastoris, an insectcell, such as, for example, Spodoptera frugiperda, or a mammalian cell,such as, for example, a COS, CHO, 293, BHK or NS0 cell.

Methods of Administration

The invention comprises methods of treatment comprising administering toa subject an effective amount of an agent of the invention. In apreferred aspect, the agent is substantially purified (e.g.,substantially free from substances that limit its effect or produceundesired side-effects). The subject is preferably an animal, e.g., suchas cows, pigs, horses, chickens, cats, dogs, etc., and is preferably amammal, and most preferably human.

Preferably, the pharmaceutical compositions of the invention areadministered to the area in need of treatment by topical administration.Topical drug delivery is the most common treatment for diseases ordisorders of the anterior segment of the eye, including, for example,corneal diseases, uveitis, and glaucoma. Topical delivery can be a saferand more convenient delivery method for patients, and can reduce therisk of many side effects observed in systemic treatment regimens.Topical administration of an angiogenesis inhibitor to the eye or corneacan be an effective treatment for treating neovascularization and/orinflammation. A preferred method of administering the pharmaceuticalcompositions of the invention to the eye is by eye drops comprising aVEGF trap.

In various preferred embodiments, the pharmaceutical compositions of theinvention are administered to the area in need of treatment bysubconjunctival administration. One preferred method of subconjunctivaladministration to the eye is by injectable formulations comprising aVEGF trap. Another preferred method of subconjunctival administration isby implantations comprising slow releasing VEGF trap.

Pharmaceutical Compositions

Pharmaceutical compositions useful in the practice of the method of theinvention include a therapeutically effective amount of an active agentwith a pharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly, inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the therapeutic is administered. Examples of suitablepharmaceutical carriers are described in “Remington's PharmaceuticalSciences” by E. W. Martin. In a preferred embodiment, the composition isformulated in accordance with routine procedures as a pharmaceuticalcomposition adapted for topical administration to human beings. Suchpharmaceutical compositions may be liquid, gel, ointment, salve, slowrelease formulations or other formulations suitable for ophthalmicadministration. The composition comprises an effective amount of VEGFantagonist and, optionally, at least one ophthalmically acceptableexcipient, wherein the excipient is able to reduce a rate of removal ofthe composition from the eye by lacrimation, such that the compositionhas an effective residence time in the eye of about 2 hours to about 24hours.

In various embodiments, compositions of the invention can comprise aliquid comprising an active agent in solution, in suspension, or both.The term “suspension” herein includes a liquid composition wherein afirst portion of the active agent is present in solution and a secondportion of the active agent is present in particulate form, insuspension in a liquid matrix. As used herein, liquid compositionsinclude gels.

Preferably the liquid composition is aqueous. Alternatively, thecomposition can take form of an ointment. In a preferred embodiment, thecomposition is an in situ gellable aqueous composition, more preferablyan in situ gellable aqueous solution. Such a composition can comprise agelling agent in a concentration effective to promote gelling uponcontact with the eye or lacrimal fluid in the exterior of the eye.Suitable gelling agents non-restrictively include thermosetting polymerssuch as tetra-substituted ethylene diamine block copolymers of ethyleneoxide and propylene oxide (e.g., poloxamine 1307); polycarbophil; andpolysaccharides such as gellan, carrageenan (e.g., kappa-carrageenan andiota-carrageenan), chitosan and alginate gums. The phrase “in situgellable” includes not only liquids of low viscosity that can form gelsupon contact with the eye or with lacrimal fluid in the exterior of theeye, but also more viscous liquids such as semi-fluid and thixotropicgels that exhibit substantially increased viscosity or gel stiffnessupon administration to the eye or area surrounding the eye.

Aqueous compositions of the invention have ophthalmically compatible pHand osmolality. Preferably these compositions incorporate means toinhibit microbial growth, for example through preparation and packagingunder sterile conditions and/or through inclusion of an antimicrobiallyeffective amount of an ophthalmically acceptable preservative. Suitablepreservatives non-restrictively include mercury-containing substancessuch as phenylmercuric salts (e.g., phenylmercuric acetate, borate andnitrate) and thimerosal; stabilized chlorine dioxide; quaternaryammonium compounds such as benzalkonium chloride, cetyltrimethylammoniumbromide and cetylpyridinium chloride; imidazolidinyl urea; parabens suchas methylparaben, ethylparaben, propylparaben and butylparaben, andsalts thereof; phenoxyethanol; chlorophenoxyethanol; phenoxypropanol;chlorobutanol; chlorocresol; phenylethyl alcohol; disodium EDTA; andsorbic acid and salts thereof.

The composition can comprise an ophthalmic depot formulation comprisingan active agent for subconjunctival administration. The ophthalmic depotformulation comprises microparticles of essentially pure active agent,e.g., the VEGF trap of SEQ ID NO:2. The microparticles comprising VEGFtrap can be embedded in a biocompatible pharmaceutically acceptablepolymer or a lipid encapsulating agent. The depot formulations may beadapted to release all of substantially all the active material over anextended period of time. The polymer or lipid matrix, if present, may beadapted to degrade sufficiently to be transported from the site ofadministration after release of all or substantially all the activeagent. The depot formulation can be liquid formulation, comprising apharmaceutical acceptable polymer and a dissolved or dispersed activeagent, Upon injection, the polymer forms a deot at the injections site,e.g. by gelifying or precipitating.

The composition can comprise a solid article that can be inserted in asuitable location in the eye, such as between the eye and eyelid or inthe conjunctival sac, where the article releases the active agent.Release from such an article is preferably to the cornea, either vialacrimal fluid that bathes the surface of the cornea, or directly to thecornea itself, with which the solid article is generally in intimatecontact. Solid articles suitable for implantation in the eye in suchfashion generally comprise polymers and can be bioerodible ornon-bioerodible. Bioerodible polymers that can be used in preparation ofocular implants carrying a VEGF trap in accordance with the presentinvention include without restriction aliphatic polyesters such aspolymers and copolymers of poly(glycolide), poly(lactide),poly(ε-caprolactone), poly(hydroxybutyrate) and poly(hydroxyvalerate),polyamino acids, polyorthoesters, polyanhydrides, aliphaticpolycarbonates and polyether lactones. Illustrative of suitablenon-bioerodible polymers are silicone elastomers.

The active agents of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

Combination Therapies

In various embodiments, the VEGF antagonists of the present inventionmay be administered in combination with one or more additional compoundsor therapies or medical procedures. For example, suitable therapeuticagents for use in combination, either alternating or simultaneously,with the VEGF-binding fusion proteins of the invention, includingtopically administered immunosuppressive agents such as corticosteroids,dexamethasone, cyclosporin A, FK506, or anti-metabolic agents, (seeBarker, N H, et al., (2000) Clin Exp Opthal 28:357-360). Other suitabletherapeutic agents for use in combination, either alternating orsimultaneously, with the VEGF antagonists of the invention may includeagents that can block the biological activity of other VEGF familymembers such as VEGF-C and VEGF-D.

Kits

The invention also provides an article of manufacture comprisingpackaging material and a pharmaceutical agent contained within thepackaging material, wherein the pharmaceutical agent comprises at leastone VEGF-specific fusion protein of the invention and wherein thepackaging material comprises a label or package insert that indicatesthat the VEGF-specific fusion protein can be used for treating eyeinjury. The kit can comprise a composition comprising a VEGF trap andone or more other components such as, for example, components to becombined prior to use either by a health care professional or by thesubject. In one embodiment, the VEGF trap is combined with one or morecomponents that can comprise, for example, a solution included in thekit to reconstitute a VEGF trap in the form of an ophthalmicalcomposition suitable for topical or subconjunctival administration to ahuman or animal. Kit components can comprise, for example, normal salinesolutions and/or solutions comprising one or more suitablepharmaceutical carriers, stabilizers, additives, or buffers. Preferablythe kit comprises instructions for treatment or administration regimensand/or instructions for preparing or reconstituting a VEGF trap for use.The instructions can be in writing on paper, on computer media of anysuitable type, as audiovisual materials including, for example, CD orDVD, or any other suitable format.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

EXAMPLES

The following example is put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the methods and compositions of the invention, and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers used (e.g., amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, molecular weight is averagemolecular weight, temperature is in degrees Centigrade, and pressure isat or near atmospheric.

Example 1 Effect of Eye Drop Administration of VEGF Trap on CornealNeovascularization

Topical administration at the cornea of a VEGF trap can effectivelyinhibit or reduce corneal neovascularization and/or inflammation of aninjured cornea. Corneal injuries, such as those sustained on repeatpenetrating keratoplasty and corneal graft procedures, can lead toinflammation and/or neovascularization of the cornea, sometimesresulting in corneal graft rejection.

A study of corneal neovascularization following suture injury in thepresence and absence of topically administered VEGF trap was conducted.VEGF trap (SEQ ID NO:2) was administered in a pharmaceutical formulationfor topical administration to the eye (i.e., VEGF trap eye drops) at pH6.0 which contained 39.4 mg/ml VEGF trap in 5 mM phosphate, 5 mM citricacid, 100 mM NaCl, and 0.0005% Polysorbate 20. Male C57BL6 mice, 8-10weeks old were used for the experiment. Each mouse underwent sutureplacement (three 10-0 nylon) in the peri-center of the right corneaunder surgical microscope. The left cornea of each mouse was used ascontrol. The mice were then placed in two groups: Each mouse in Group Ireceived one normal saline eyedrop (4 microliters) (μl) on the rightsutured cornea three times a day for eight days. Each mouse in Group IIreceived one VEGF trap eye drop (4 μl) on the right sutured cornea threetimes a day for eight days. The saline eye drops contained 0.9% sodiumchloride, Injection, USP, pH 5.6 (4.5-7.0) (Lot #01-172-JT, AbbottLaboratories, North Chicago, Ill.), where each 100 mL of the saline eyedrops contained 900 mg NaCl in water. The VEGF trap eye drops contained157.6 μg of VEGF trap/drop (4 μl/drop). On the eighth day,fluorescinated lectin (Lycopersicon esculentum) was injectedintravenously to label the vasculature. Corneal flat-mounts were thenprepared and the area of neovascularization measured.

The results demonstrate that sutured corneas receiving VEGF trap eyedrops exhibited little or no corneal neovascularization relative to thetotal vascular area of the unsutured, untreated controls. In contrast,sutured mice treated with only normal saline exhibited markedneovascularization, showing about a four-fold increase in cornealvascular area. This study demonstrates that topical administration ofVEGF trap almost completely suppresses corneal neovascularizationfollowing corneal suture placement. Topical administration of VEGF trapalso effectively reduced inflammation induced by corneal injury. Thus,topical administration of a VEGF trap is a highly effective method ofreducing or eliminating neovascularization and inflammation due tocorneal disease, disorder, or injury.

Example 2 Effect of Subconjunctival Administration of VEGF Trap onCorneal Neovascularization in Rats

The efficacy of subconjunctival delivery of a VEGF trap on cornealinjury in the mammal was evaluated by observing the effects ofsubconjunctival delivery of a VEGF trap in suture-injured rat corneas.Neovascularization following corneal suture placement was measured inthe presence and in the absence of subconjunctival delivery of a VEGFtrap. Male Sprague-Dawley rats (200-250 g) underwent intrastromal sutureplacement (three 10-0 nylon sutures/eye) in the peri-center of thecornea. VEGF trap (SEQ ID NO:2) was injected subconjunctivally at doseof 200 μg in 50 μl per eye per day at day 0, day 2, and day 5 followingsuture placement. As a control, 50 μl of vehicle, lacking VEGF trap, wasinjected per eye at day 0, day 2, and day 5 following suture placement.On day 8 and day 12, corneal neovascularization was evaluated in cornealflat mounts. Representative corneas taken on day 8 also were fixed andembedded in paraffin. Sections were stained with hematoxylin and eosin.Leukocyte infiltration and corneal thickness were measured as indices ofinflammation and edema, respectively.

The results showed that corneal flat mounts on day 8 from rats treatedwith subconjunctival administration of vehicle only displayedsignificant neovascularization in the vicinity of the corneal injury(i.e., in the vicinity of the sutures). Neovascularization developedfrom the limbal vessels to the three sutures in the peri-central cornea.Corneal flat mounts on day 8 from rats treated with subconjunctivaladministration of VEGF displayed very little neovascularizationreflecting almost total inhibition of neovascularization.

Even seven days following the final subconjunctival injection of VEGFtrap (i.e., even at day 12), corneal flat mounts displayed virtually noneovascularization. 12 day corneal flat mounts were made of sutured ratstreated with subconjunctival administration of vehicle only, or VEGFtrap. The sutured corneas treated with VEGF trap exhibited significantinhibition of neovascular proliferation, whereas corneas treated withvehicle only were still at peak neovascularization.

Histological analysis of corneas on day 8 revealed that suture-injuredrats that received vehicle only displayed multiple layers of neovascularformation in the cornea, extensive leukocyte (inflammatory) cellinfiltration of the cornea, and significant corneal thickening. Incontrast, day 8 corneas of rats treated with subconjunctival injectionof VEGF trap displayed substantially less infiltration of inflammatorycells, little or no corneal neovascularization, an a reduction incorneal thickening as compared to untreated injured rat corneas.

Example 3 Effect of Subconjunctival Administration of VEGF Trap onCorneal Neovascularization in Mice

The efficacy of subconjunctival injection of the VEGF trap onneovascularization induced by corneal suture injury was evaluatedfurther in mice using three dosing regimens. Male C57BL6 mice (8-10weeks old) underwent suture placement (three 10-0 nylon sutures/eye) inthe peri-central cornea. VEGF trap (SEQ ID NO:2) was injectedsubconjunctivally using the following dosing regimens: 40 μg on day 0,day 2, and day 5 following suture placement; 10 μg on day 0, day 2, andday 5 following suture placement; or 10 μg on day 0 following sutureplacement.

Using the above dosing regimens, mice were injected subconjunctivallywith VEGF trap or vehicle only (no VEGF trap) and corneal flat mountstaken on day 9 were prepared and evaluated as described above.

The effect on day 9 of a subconjunctival VEGF trap dosing regimen of 40micrograms on each of days 0, 2, and 5 following suture placement isshown in FIG. 1A. This dosing regimen effectively inhibitsneovascularization, as measured by an increase in percent cornealvascular area (about 5% in uninjured, untreated mice, data not shown).The effect on day 9 of a subconjunctival VEGF trap dosing regimen of 10μg on each of days 0, 2, and 5 following suture placement is shown inFIG. 1B which reveals that this dosing regimen also results in adecrease in corneal neovascular area compared with corneas treated withvehicle only (p<0.01).

The effect on day 9 of a single subconjunctival VEGF trap dose of 10 μgon the day of suture placement (FIG. 2) reveals that even a singlesubconjunctival dose of VEGF on the day of suturing can reduceneovascularization in the cornea by about half (p<0.05).

Example 4 Effect of Systemic or Subconjunctival Administration of VEGFTrap on Corneal Neovascularization, Inflammation and Edema

A study was conducted to determine the minimally effective dose of VEGFtrap (SEQ ID NO:2) administered by systemic (subcutaneous, SC)injection, or local (subconjunctival, SubC) injection on cornealneovascularization, inflammation and edema. Male Sprague Dawley rats ofapproximately 250 g body weight received 3 loop sutures in the rightcornea and the left eye was used as control. Six experimental groups(n=5 per group) received 3 SC or 3 SubC injections of VEGF trap (SEQ IDNO:2) at day 0, 3 and 6, at the following concentrations: 25 μg/50 μl,50 μg/50 μl, or 100 μg/50 μl. The subconjunctival formulation was 4.92mg/ml VEGF trap protein (SEQ ID NO:2) (Lot # VGTF00002T) in 5 mMphosphate, 5 mM citrate, 50 mM NaCl, 5% (w/v) sucrose, 0.1% polysorbate20 (Tween=20), pH 6.0. Samples were harvested on day 9 for evaluation ofcorneal blood vessels in flat mounts stained with fluoresceinatedconcanavalin A. Evaluation included in-life slit-lamp examination andquantification of corneal neovascularization. Corneal neovascularizationwas quantified in concanavalin-stained flat mounts by measuring theincrease in length of corneal blood vessels from the corneal limbus. Theresults, shown in FIG. 3, show that blood vessel length wassignificantly greater in the systemically injected (SC) animals relativeto the locally injected (SubC) animals (p<0.001).

Example 5 Effect of Subconjunctival Administration of VEGF Trap onCorneal Neovascularization, Inflammation and Edema

A study was conducted as outlined above in male Sprague Dawley ratsinjected subconjunctivally on day 0, 3, and 6 with normal saline, 5 μg,25 μg, or 100 μg of VEGF trap (SEQ ID NO:2). Eyes were harvested andexamined as described above. FIG. 4 shows quantification of blood vessellength for each experimental group. Compared to the normal salinetreated eyes, subconjunctival administration of VEGF trap significantlyinhibited corneal neovascularization at all doses tested. The extent ofinhibition was dose dependent (82%, 88% and 97% inhibition for 5 μg, 25μg, and 100 μg, respectively).

Example 6 Quantification of Corneal Edema in Animals Treated withSubconjunctival or Systemic Injections of VEGF Trap

A study was conducted as described above. Male Sprague Dawley rats wereinjected subcutaneously with 25 or 100 μg VEGF trap or subconjunctivallywith normal saline, 5 μg, 25 μg, or 100 μg VEGF trap. Injections wereadministered at day 0, 3 and 6. FIG. 5 shows quantification of cornealedema as evidenced by increased corneal thickness relative to thethickness of the cornea in normal, uninjured control. Compared to thesaline treated group, subconjunctival administration of VEGF trapsignificantly reduced corneal thickness following suture injury in adose dependent manner (28% to 56% reduction of excess cornealthickness). FIG. 6 shows the percent reduction of edema achieved with SCor SubC administration of the VEGF trap relative to control groups.

Example 7 Effect of Eye Drop Administration of VEGF Trap on CornealNeovascularization

Male C57BL6 mice, 8-10 weeks old, received suture placement (three 10-0nylon sutures) in the peri-central right cornea. The topical eye dropformulation was 103.06 mg/ml VEGF trap protein (SEQ ID NO:2) (Lot#C04002M400) in 5 mM phosphate, 5 mM citric acid, 100 mM NaCl, 0.0005%polysorbate 20, pH 6.0. The animals were treated with eye dropstopically applied to the sutured injured eye of each animal containingeither vehicle or VEGF trap (412 μg/4 μl) three times per day for 9days. Uninjured, untreated eyes served as controls. On day 9, animalsreceived an intravenous injection of fluoresceinated lectin(Lycopersicon esculentum) and corneal flat-mounts were prepared forquantification of corneal neovascularization. Blood samples were takenand free VEGF trap levels measured by ELISA.

The effect of VEGF trap administered in eye drops is shown in FIG. 7.Topical administration of VEGF trap inhibited corneal neovascularizationby 97% inhibition following suture placement at 9 days. Topical VEGFtrap treatment also markedly reduced inflammation and edema induced bycorneal injury.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof.

1.-14. (canceled)
 15. An ophthalmic composition comprising a VEGFantagonist in a pharmaceutically acceptable carrier, wherein the VEGFantagonist is VEGFR1R2-FcΔC1(a).
 16. The ophthalmic composition of claim15, which is a liquid, gel, ointment, salve, or sustained releaseformulation.